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Principal Investigator: Andre LaRochelle
Organization: NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
Fiscal Year: 2023
Award: $1,332,597
Funding agency: National Heart Lung and Blood Institute
Objective 1: Develop a scalable culture system for hematopoietic differentiation of hiPSCs
To address the limitations of current iPSC differentiation approaches, we initially sought to develop a chemically-defined and scalable hematopoietic differentiation protocol requiring no replating, EB formation or co-culture on stromal elements. Through systematic optimization steps, we established a simple 21-day monolayer-based culture system that recapitulates ex vivo the emergence of human hematopoiesis. Under culture conditions that favored mesodermal specification of human iPSCs (Day 0 to 3), an adherent monolayer rapidly formed. With the subsequent addition of hematopoietic cytokines (Day 3 to 21), hematopoietic clusters emerged from the monolayer before their eventual release in the supernatant fraction.
We systematically characterized cells arising from this system by harvesting supernatant and monolayer populations at regular intervals between day 5 and 21 of differentiation. Within the supernatant, hematopoietic cells (CD43+CD45+/-) underwent sequential development with features of primitive wave hematopoiesis (peak at day 7), definitive multilineage HSPCs with potent colony formation activity in vitro but limited engraftment potential in vivo (peak at day 12), and definitive erythroid-committed progenitors expressing adult-type globin chains (peak at day 17 to 21). To understand the possible causes underpinning the absence of engraftable HSPCs in this system, we examined the cellular constituents of the CD43-CD45- non-hematopoietic fraction that supports hematopoiesis during differentiation. We first identified a prevalent population of mesenchymal cells throughout differentiation. Perivascular mesenchymal cells are known to interact with HSPCs and maintain their activity in the adult BM niche, but their role in promoting definitive HSPC development during ontogeny has not been demonstrated. Importantly, arterial HE was largely absent within the supportive monolayer. Thus, in keeping with prevailing arterial-specification models, we deduced that the adherent monolayer was likely inadequate to support the generation of bona fide engrafting HSCs in culture Ruiz et al., Stem Cell Res 2019.
Objective 2. Optimize iPSC differentiation conditions to promote arterial HE formation
To augment the production of arterial HE during iPSC differentiation, we postulated that simultaneous activation of key signaling pathways independently shown to control arterial fate during vascular development, including WNT/-catenin, activin/nodal/TGF, and mitogen-activated protein kinase (MAPK)/ERK pathways, might provide a synergy sufficient to further instruct definitive hematopoiesis with engraftment potential from human iPSCs. To test this possibility, we supplemented the culture medium with the WNT/-catenin agonist CHIR99021 (CHIR) and nodal/activin/TGF inhibitor SB431542 (SB) at day 2 of iPSC differentiation and activated MAPK/ERK signaling with LY294002 (LY) from day 3 to day 6 of culture. Control cultures contained no CHIR/SB/LY (non-treated), or were supplemented with CHIR/SB or LY only. Addition of CHIR/SB/LY led to a marked increase in percentages and numbers of CD144+CD34hiCD73-CD184+ arterial HE, peaking at day 5 of differentiation. We next investigated whether this early increase in arterial HE formation observed in the presence of CHIR/SB/LY influenced hematopoietic development. Addition of CHIR/SB/LY decreased overall CD43+/-CD45+ hematopoietic cell numbers but a notable rise in percentages of phenotypically defined definitive HSPCs (CD34+CD45RA-CD90+) was observed within the hematopoietic population at day 12 of differentiation compared to controls. In CFU assays, the frequency of progenitors with multilineage differentiation capacity was similar between control groups but significantly increased in the CHIR/SB/LY group. To further assess the self-renewal and differentiation capacity of iPSC-derived hematopoietic CD34+ cells, colonies derived from the first round of CFU plating were pooled and replated in secondary CFU assays. Notably, we observed a 3-fold increase in total CFU numbers from CD34+ cells derived from CHIR/SB/LY cultures compared to control groups. However, these cells did not sustain long-term hematopoietic engraftment after transplantation into NSG mouse recipients, indicating that additional revisions to this system are required Li et al., Stem Cells 2023.
Objective 3. Optimize iPSC differentiation for the generation of engraftable HSPCs
While activation of an arterial program is required for HSC induction ex vivo, our data suggest that current culture conditions fail to modulate other critical molecular programs uncoupled from arterial development. Three approaches are explored for the generation of engraftable HSPCs Ding et al., Cells 2023.
Ectopic activation of master transcription factors. Using highly purified populations of human HSCs, we have combined high-throughput CHIP-seq and ATAC-seq methods and established bioinformatics pipelines to uncover HSC-specific SEs and their putative target TFs. In ongoing experiments, we are enforcing transient expression of the assembled library of master TFs using lipid nanoparticle (LNP)-based transfer of TF mRNA at various stages of iPSC differentiation. In parallel, a multiplexed CRISPRa approach is exploited for direct activation of SEs driving expression of master TFs defining HSC identify. To evaluate functionality of the differentiated cell product, we are evaluating transcriptional similarity between iPSC-derived HSPCs and distinct native cell types at various stages of human hematopoietic maturation. The most promising findings will be confirmed by gold-standard xenotransplant assays.
Inhibition of epigenetic regulators. Emerging evidence indicates that acquisition of HSC multipotency and engraftment potential during embryonic development is restricted by a distinct epigenetic barrier. Recent proof-of-concept studies demonstrated that transient inhibition of key epigenetic factors at early stages of differentiation promoted multi-lymphoid output from human iPSCs in vitro and enabled rapid maturation of newly born neurons in an iPSC differentiation model of neural progenitor cells. Building on these observations, we are exploring pharmacologic inhibition of the core subunits EZH1 and EZH2 of PRC2, a central Polycomb repressor complex involved in transcriptional repression during hematopoietic development, as well as other epigenetic factors (e.g., EHMT1/2, DOT1L) shown to limit temporal unfolding of maturation programs in pluripotent stem cell culture.
Ectopic expression of homing receptors. Efficient homing, retention, and survival within specialized niches of the recipient marrow immediately after transplantation constitute a sine qua non for long-term engraftment of HSCs produced in vitro. An early transplantation failure of iPSC-derived HSPCs was uncovered after direct intra-femoral injection in xenograft murine models. The inability of these cells to home and survive after transplantation was attributed to limited expression of the CXCR4 chemokine receptor, a well-known mediator of HSPC homing within the BM niche in vivo. Lentiviral vector(LV)-induced expression of CXCR4 in iPSC-derived HPSCs improved short-term marrow retention but was insufficient to confer sustained engraftment. To expand our understanding of the functional connectivity between BM niche and iPSC-derived HSPCs for sustained engraftment, we are optimizing the timing/dosage of CXCR4 expression during iPSC differentiation and enforcing expression of alternative surface molecules implicated in cellular adhesion (e.g., integrins, cadherins, selectins, tetraspanins) by stage-specific transduction with integrating LV vectors bearing a unique homing receptor open reading frame.
Terms: <21+ years old><ATAC sequencing><ATAC-seq><ATACseq><Activins><Address><Adult><Adult Human><Agonist><Animals><Aorta><Arteries><Assay><Autologous><Automobile Driving><B220><Basal Transcription Factor><Basal transcription factor genes><Binding><Bioassay><Biologic Assays><Biological Assay><Blood><Blood Precursor Cell><Blood Reticuloendothelial System><Blood Vessels><CD34><CD34 gene><CD45><CRISPR activation><CRISPR activator><CRISPR based activation><CRISPR gene activation><CRISPR transcription activation><CRISPR transcriptional activation><CRISPR-Cas-9-mediated gene activation><CRISPR-based gene activation><CRISPR-dCAS9 Activator><CRISPR-mediated transcriptional activation><CRISPR/CAS9 activation><CRISPR/CAS9 gene activation><CRISPR/dCas9 activation><CRISPR/dCas9-based transcriptional activation><CRISPRa><CXC-R4><CXCR-4><CXCR4><CXCR4 gene><Cadherins><Cell Adhesion><Cell Body><Cell Communication and Signaling><Cell Count><Cell Culture Techniques><Cell Differentiation><Cell 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Growth><Generations><Genes><Genetic Transcription><Globin><Gonadal structure><Growth><HM89><HPCA1><HSC Specification><HSY3RR><Harvest><Hematopoiesis><Hematopoietic><Hematopoietic Cellular Control Mechanisms><Hematopoietic Cytokine><Hematopoietic Progenitor Cells><Hematopoietic Stem Cell Specification><Hematopoietic stem cells><Heterograft><Heterologous Transplantation><Home><Homing><Human><IL-3><IL-3 Gene><IL3><IL3 Gene><IL3 Protein><Immune><Immunes><In Vitro><Injections><Integrins><Integrins Extracellular Matrix><Intracellular Communication and Signaling><Investigation><KMT6><KMT6A><LAP3><LCR1><LESTR><LY5><Lentiviral Vector><Lentivirus Vector><Libraries><Lymphoid><MAP kinase><Marrow><Mast-Cell Colony-Stimulating Factor><Mediator><Mesenchymal><Mesonephric structure><Mesonephros><Messenger RNA><Methods><Mice><Mice Mammals><Mitogen-Activated Protein Kinases><Modeling><Modern Man><Molecular><Molecular Fingerprinting><Molecular Interaction><Molecular Profiling><Morphology><Murine><Mus><NPY3R><NPYR><NPYRL><NPYY3R><Nerve Cells><Nerve Unit><Neural Cell><Neural Stem Cell><Neurocyte><Neurons><Nodal><ORFs><Oncogenic><Open Reading Frames><Output><P-CSF><P-Cell Stimulating Factor><PTPRC><PTPRC gene><Pathway interactions><Patients><Phase Transition><Phenotype><Pluripotent Stem Cells><Polycomb><Population><Production><Progenitor Cell Transplantation><Progenitor Cells><Protein Coding Region><Publishing><RNA Expression><Receptor Protein><Repressor Proteins><Role><Selectins><Self Assessment><Signal Pathway><Signal Transduction><Signal Transduction Systems><Signaling><Specific qualifier value><Specified><Stem Cell Development><Stem Cell Transplantation><Stem cell transplant><Strains Cell Lines><Stromal Cells><Surface><System><T200><Testing><Tissue Growth><Transcription><Transcription Factor Proto-Oncogene><Transcription Repression><Transcription factor genes><Transcriptional Repression><Transplantation><Vascular Endothelium><Wolffian 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tissue><iPS><iPSC><iPSCs><improved><in vitro activity><in vivo><induced pluripotent cell><induced pluripotent stem cell><inducible pluripotent stem cell><inhibitor><lentiviral integration><lentivirus integration><lipid based nanoparticle><lipid nanoparticle><mRNA><molecular profile><molecular signature><monolayer><mouse model><murine model><nerve stem cell><neural model><neural precursor><neural precursor cell><neural progenitor><neural progenitor cells><neuron progenitors><neuronal><neuronal progenitor><neuronal progenitor cells><neuronal stem cells><neuroprogenitor><ontogeny><p-Globin><pathway><pharmacologic><progenitor><progenitor cell development><programs><receptor><repressor complex><self-renew><self-renewal><social role><stem cell differentiation><stem cells><synergism><tool development><transcription factor><translation strategy><translational approach><translational strategy><transplant><vascular><vector><vector induced><xeno-transplant><xeno-transplantation>